Human cervical cancer radiotherapy resistant cell strain as well as construction method and application thereof

Abstract

The invention discloses a human cervical cancer radiotherapy resistant cell strain as well as a construction method and application thereof, and belongs to the technical field of tumor radiation biology. In order to solve the technical bottlenecks of clinical disjunction, phenotype instability and single evaluation system of an existing cervical cancer cell radiation resistance model, progressive accumulative irradiation (40 times * 2Gy, the total dose is 80Gy) is performed on human cervical cancer ME180 cells, a clinical radical radiotherapy process is simulated, and a cervical cancer radiation-tolerant strain ME180-R is constructed. In the method, a maintenance irradiation strategy of 2Gy per week is innovatively introduced, so that the radiation resistance phenotype of the cell strain can be stably passed for at least 10 generations. According to the method, a quantitative evaluation system containing multi-dimensional parameters such as D0, Dq and SF2 is established at the same time. The cell strain and the construction method thereof provide reliable and stable preclinical models and tools for transformation medical research such as cervical cancer radiotherapy sensitizer screening, radiation resistance mechanism analysis, individualized radiotherapy scheme optimization and the like.

Description

Technical Field

[0001] This invention belongs to the field of tumor radiobiology technology, specifically relating to a human cervical cancer radiotherapy resistant cell line, its construction method, and its application. Background Technology

[0002] Cervical cancer is the fourth most common malignant tumor among women worldwide, and radiotherapy (including external beam radiation and brachytherapy) is widely used in clinical practice as a core means of radical treatment. However, approximately 30% of patients develop radiotherapy resistance due to acquired tumor cells, resulting in local recurrence or distant metastasis, leading to a 5-year survival rate of less than 20%. Therefore, in-depth research into the underlying mechanisms of radiotherapy resistance in cervical cancer and the establishment of reliable preclinical research models are crucial to overcoming the bottlenecks in cervical cancer treatment and improving patient prognosis.

[0003] Currently, the mainstream methods for constructing radiation-resistant cell lines are mainly divided into two categories: one is the short-term high-dose induction method, which uses a single ultra-high dose irradiation (usually 10~20Gy) to achieve resistance induction by relying on acute radiation injury pressure. It can be completed in just 1~2 irradiations and has a short cycle (≤1 week); the other is the limited fractionated irradiation method, which uses multiple irradiation modes to simulate fractionated radiotherapy, but the cumulative dose is low (usually ≤40Gy) and does not reach the equivalent dose (70~80Gy) required for clinical radical cure.

[0004] However, existing methods for constructing radiation-resistant cell models have fundamental flaws, the core problem being a severe disconnect from clinical reality. The mainstream protocols employing single ultra-high-dose irradiation (10-20 Gy) or low-cumulative-dose fractionated irradiation (≤40 Gy) deviate significantly from the standard protocol for radical radiotherapy of cervical cancer (25-40 fractions × 1.8-2 Gy, total dose 70-80 Gy). This difference in dose fractionation leads to a deviation from clinical reality in cell death mechanisms: short-term high-dose induction primarily results in necrosis, failing to simulate the crucial process of gradual cellular adaptation in fractionated radiotherapy; while insufficient cumulative dose makes it difficult to activate the metabolic reprogramming and immune escape mechanisms of tumor cells under long-term treatment stress, rendering the constructed models unable to truly reflect the biological essence of patients' radiation resistance.

[0005] Another major drawback of existing technologies is the high instability of resistance phenotypes. In most reported resistant strains, resistance significantly degrades within 5-10 passages after irradiation cessation. The root cause lies in the lack of a design that maintains sustained selection pressure; during routine passages, the expression levels of radiation damage response pathways in cells rapidly decline back to baseline, rendering the model unusable for research. This degradation severely restricts the systematic exploration of radiotherapy resistance mechanisms and the long-term efficacy evaluation of radiosensitizers.

[0006] Furthermore, the one-sidedness and low standardization of existing evaluation systems further limit the translational application of models. Current research largely relies on a single survival rate indicator, neglecting the quantitative analysis of key parameters of radiosensitivity: the lack of mean lethal dose (D0 value) makes it impossible to accurately assess the intrinsic radiosensitivity of cells; the quasi-threshold dose (D... q The lack of measurement of the radiotherapy resistance value makes it difficult to quantify the ability to repair sublethal damage; and the survival score (SF2), as the gold standard for clinical fractionated radiotherapy sensitivity, is not widely used. These deficiencies in the evaluation system lead to a lack of comparability of data between different studies, severely hindering in-depth analysis of radiotherapy resistance mechanisms. Summary of the Invention

[0007] To address the limitations of existing technologies, this invention aims to provide a human cervical cancer radiotherapy-resistant cell line, its construction method, and its application.

[0008] The technical solution adopted in this invention is as follows: A human cervical cancer radiotherapy resistant cell line ME180-R was deposited on August 20, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 66838, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Furthermore, the human cervical cancer radioresistant cell line ME180-R is a cell and / or its progeny cells with a stable radioresistant phenotype obtained by progressive cumulative X-ray irradiation of human cervical cancer cells ME180; wherein the stable radioresistant phenotype can be stably passaged for at least 10 generations under maintenance irradiation of 2 Gy per week.

[0009] The above-mentioned method for constructing the human cervical cancer radiotherapy-resistant cell line ME180-R and maintaining its stable radioresistance phenotype includes the following steps: S1: Initial cell culture: Obtain and culture human cervical cancer cells ME180; S2: Induction irradiation: When the human cervical cancer cells ME180 are in the exponential growth phase, fractionated X-ray irradiation is started, with a single irradiation dose of 2 Gy every 72 hours, for a cumulative 40 irradiations, and a total dose of 80 Gy is obtained to obtain the human cervical cancer radiotherapy resistant cell line ME180-R. S3: Maintenance irradiation: After the human cervical cancer radioresistant cell line ME180-R obtained in step S2 is passaged for 2-3 generations, X-ray maintenance irradiation is started at a frequency of 2 Gy once a week to maintain the stability of its radioresistant phenotype. In steps S2 and S3, the irradiation is performed using 6MV X-rays generated by a linear accelerator at a dose rate of 300 cGy / min. During the irradiation and culture in steps S2 and / or S3, cell digestion and passage are performed when the cell confluence reaches 90%. The method further includes a step of evaluating the radiosensitivity of the constructed cell lines in multiple dimensions to quantify their radioresistance phenotype. This evaluation includes determining their mean lethal dose D0 and quasi-threshold dose D0. q Survival fraction (SF2) and radiation resistance factor.

[0010] The above-mentioned human cervical cancer radiotherapy resistant cell line ME180-R is used in the preparation of preclinical research models, reagents or kits for screening and / or evaluating cervical cancer radiosensitizers.

[0011] The above-mentioned human cervical cancer radiotherapy resistant cell line ME180-R is used in the preparation of preclinical research models, reagents or kits for studying the molecular mechanisms of cell survival regulation related to radiotherapy resistance in cervical cancer.

[0012] A kit for assessing the radiosensitivity of cells, the kit comprising the aforementioned human cervical cancer radiotherapy-resistant cell line ME180-R.

[0013] A preclinical evaluation system for screening radiosensitizers, the system comprising the aforementioned human cervical cancer radiotherapy-resistant cell line ME180-R.

[0014] The beneficial effects of this invention are as follows: (1) A progressive fractionated irradiation regimen with a total dose of 80 Gy (40 fractions × 2 Gy) was adopted to accurately simulate the clinical radical radiotherapy process of cervical cancer. This ensured that the cells gradually activated adaptive biological mechanisms such as metabolic reprogramming and DNA repair enhancement, which are similar to the patient's radiotherapy resistance, during the induction period of up to 4 months, thus greatly improving the clinical relevance of the model.

[0015] (2) An innovative strategy of maintaining irradiation at 2 Gy per week after construction was introduced. By continuously applying selection pressure, the expression of radiation damage response pathways was suppressed and the radiation resistance phenotype of ME180-R cell line was successfully stably passaged for at least 10 generations (survival fraction SF2 fluctuation <3%), which solved the key defect of rapid phenotype degradation within 5 to 10 generations after irradiation was stopped in the existing model.

[0016] (3) The constructed ME180-R cell line showed significant radioresistance, with an average lethal dose D0 of 3.45 Gy (62.7% higher than the parent cell line), a survival fraction SF2 of 0.647 (44.1% higher than the parent cell line), and a radioresistance fold of 1.44. Furthermore, the quasi-threshold dose D0 was significantly lower than the parent cell line. q The increase in the survival curve (widening of the shoulder area) clearly validates the radiotherapy resistance, providing a typical model for related mechanism research.

[0017] (4) Integrating mean lethal dose D0 and quasi-threshold dose D q By incorporating multiple key parameters such as survival fraction (SF2) and radiation resistance factor, a standardized evaluation system was established that goes beyond a single survival rate indicator. This enables quantitative analysis of radiosensitivity, ensures data comparability between different studies, and provides a reliable tool for in-depth analysis of resistance mechanisms.

[0018] (5) This standardized cell line model and supporting technology can be directly applied to high-throughput screening of radiosensitizers, in-depth research on resistance mechanisms such as DNA damage repair, and optimization of individualized radiotherapy regimens, filling the gap in the standardized toolchain for cervical cancer radioresistance research and having clear clinical translational value. Attached Figure Description

[0019] Figure 1 , Figure 2 Colony formation results and corresponding survival curves from two independent experiments.

[0020] Figure 3 : Figure 1 and Figure 2 The fitted curve of the average value of the two experimental data.

[0021] Figure 4 Bottlenecks in existing technologies. Detailed Implementation

[0022] To further understand the present invention, the technical solutions of the present invention will be clearly and completely described below in conjunction with embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] Example 1: Construction, phenotype maintenance, and performance validation of the human cervical cancer radiotherapy resistant cell line ME180-R I. Construction and Phenotypic Maintenance of ME180-R Cell Line S1: Initiation of Cell Culture and Preparation Human cervical cancer cells ME180 (ATCC accession number: HTB-33) were obtained and routinely cultured in McCoy's 5A complete medium containing 10% inactivated fetal bovine serum at 37°C in a 5% CO2 incubator. The cells were then cultured at a rate of 4 × 10⁻⁶ cells / year. 5 The cells were seeded at a density of 1 cell per T25 culture flask, with the culture medium replaced with fresh medium every 48 hours. Once the cells reached the exponential growth phase (60%–70% confluence), they were used for subsequent irradiation induction. A control group was also set up, where parental ME180 cells received no irradiation treatment and underwent routine culture and passage simultaneously with the experimental group.

[0024] S2: Progressive cumulative induced irradiation ME180 cells in the exponential growth phase (60%–70% confluence) of S1 were irradiated using an ElektaSynergy linear accelerator in Sweden. Irradiation parameters were set to 6MV X-rays at a dose rate of 300 cGy / min; fractionated irradiation was used, with a single dose of 2 Gy administered every 72 hours, for a total of 40 fractions and a total dose of 80 Gy (the induction period was approximately 4 months). During irradiation, the cells continued to be cultured routinely in McCoy's 5A complete medium containing 10% inactivated fetal bovine serum at 37°C in a 5% CO2 incubator. When confluence reached 90%, the cells were passaged using trypsin digestion. After all irradiation was completed, the human cervical cancer radiotherapy-resistant cell line ME180-R was obtained.

[0025] The human cervical cancer radiotherapy resistant cell line ME180-R was deposited on August 20, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 66838, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0026] S3: Resistance to phenotype stabilization and maintenance of irradiation The radioresistant human cervical cancer cell line ME180-R, obtained from S2, was routinely passaged for 2-3 generations in McCoy's 5A complete medium containing 10% inactivated fetal bovine serum at 37°C in a 5% CO2 incubator (trypsin digestion was performed when confluence reached 90%). Then, a maintenance irradiation program was initiated. Maintenance irradiation was performed using the same equipment and parameters as S2 (ElektaSynergy linear accelerator, 6MV X-rays, dose rate 300 cGy / min). During routine passage, X-ray irradiation of 2 Gy was interspersed weekly. This continuous low-dose irradiation served as selection pressure to stably maintain the radioresistant phenotype of the human cervical cancer radioresistant cell line ME180-R.

[0027] II. Verification and Results of Radiation Resistance Performance 1. Methods for determining radiosensitivity (colony formation experiment) Parental cells (ME180) and radioresistant cells (ME180-R) in the exponential growth phase were selected and digested with trypsin until complete cell detachment. Digestion was then stopped, and the cells were gently pipetted to prepare a single-cell suspension. A cell counting chamber was used to accurately count the cells to ensure consistent cell concentrations. Equal numbers of cells were seeded into six-well plates at a density suitable for colony formation, with six dose gradients: 0 Gy (blank control), 2 Gy, 4 Gy, 6 Gy, 8 Gy, and 10 Gy. Each dose gradient had three replicates. After seeding, the six-well plates were incubated overnight in a 5% CO2, 37°C incubator to allow for full cell adhesion. The next day, the six-well plates were removed, and each well was irradiated individually using a Swedish Elekta Synergy linear accelerator (irradiation parameters set to 6 MV X-rays, dose rate 300 cGy / min) according to the preset dose gradient. Immediately after irradiation, the six-well plates were returned to the same culture environment for continued incubation, avoiding frequent movement to ensure a stable cell growth environment. Terminate the culture when visible cell clones are observed in the wells. Carefully discard the culture medium from each well. Slowly add PBS buffer along the well wall and gently wash the cell surface three times. Discard the buffer and add 4% paraformaldehyde solution to each well. Incubate at room temperature to fix the cells and preserve their morphology. Then discard the fixative and gently wash three more times with PBS buffer to remove any residual fixative. Add crystal violet staining solution to each well and incubate at room temperature in the dark to allow the cell clones to stain thoroughly. After staining, discard the staining solution and slowly rinse the six-well plate with deionized water until all residual purple staining is removed. Allow the plate to air dry at room temperature. Finally, count the number of colonies formed in each group under a microscope (each colony must contain ≥50 cells). Repeat the above experimental steps independently twice.

[0028] 2. Data Calculation Basic parameter calculation: Inoculation rate: Inoculation rate = [colony formation number (0 Gy) / cell seeding number (0 Gy)] × 100% Survival score (SF): Survival score (SF) = number of colonies formed (XGy) / [(number of seeded cells (XGy) × inoculation rate)], where the survival score at a dose of 2Gy is recorded as SF2, which serves as the core evaluation indicator for clinical fractionated radiotherapy sensitivity.

[0029] Definition and calculation of key parameters for radiosensitivity: Mean lethal dose (D0): Represents the radiosensitivity of the cell population, i.e., the radiation dose required to kill 63% of the cells. The smaller the D0 value, the higher the cell radiosensitivity. Obtained by fitting survival curves using a click-multi-target model.

[0030] Precision threshold dose (D) q ): This represents the shoulder width of the survival curve, reflecting the cell's ability to repair non-lethal damage. Within this dosage range, cells can repair non-lethal damage. Dq The higher the value, the greater the dose required to induce exponential cell death, and the stronger the cell repair capacity. This value was obtained by fitting survival curves using a click-to-multi-target model.

[0031] Radiation resistance factor: used to quantify the degree of enhanced survival ability of resistant cells relative to parental cells. The calculation formula is: Radiation resistance factor = SF2 (resistant cells) / SF2 (parental cells).

[0032] Survival curve plotting and differential analysis: A single-target multi-target model was used to fit the survival score data from two independent repeated experiments. Figure 1 , Figure 2 The figures show the results of colony formation experiments and the corresponding survival curves from two independent experiments. Figure 3 The curve is fitted to the average of the two experimental data. Based on Figure 3 By comparing the survival curve morphology, D0 value, and D... q The values ​​of SF2 and radiation resistance factor were used to analyze the differences in radioresistance between the two groups of cells.

[0033] 3. Experimental Results and Analysis The core evaluation parameters obtained from the above experiments are shown in the table below:

[0034] The results showed that the human cervical cancer radiotherapy resistant cell line ME180-R had significantly higher D0 and Dq values ​​than its parental cell line ME180, demonstrating enhanced intrinsic radiation sensitivity and sublethal damage repair capabilities; its SF2 value was also significantly increased, with a radioresistance fold of 1.44, confirming that it possessed a stable and significant radioresistance phenotype.

[0035] 4. Phenotypic stability verification To further confirm that the radioresistance phenotype of the human cervical cancer radiotherapy-resistant cell line ME180-R can remain stable during long-term passage, ME180-R cells were continuously passaged under S3 maintenance irradiation conditions, and key radiosensitivity indicators at critical passages were tracked and verified. The specific verification method is as follows: ME180-R cells were continuously passaged under the S3 maintenance irradiation conditions. At the 10th passage, the colony formation experiment was repeated according to the previously described radiosensitivity determination method (colony formation experiment), and the SF2 value of the cells was measured. The results showed that the SF2 value of the human cervical cancer radiotherapy-resistant cell line ME180-R remained stably maintained within the range of 0.63~0.65 at the 10th passage, with a coefficient of variation of less than 3%. This result confirms that the maintenance irradiation method of the present invention can effectively maintain the long-term stability of the radioresistance phenotype of the human cervical cancer radiotherapy-resistant cell line ME180-R.

[0036] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. A human cervical cancer radioresistant cell line ME180-R, characterized in that: The human cervical cancer radiotherapy resistant cell line ME180-R was deposited on August 20, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 66838, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. The human cervical cancer radioresistant cell line ME180-R according to claim 1, characterized in that: The human cervical cancer radioresistant cell line ME180-R is obtained by inducing human cervical cancer cells ME180 through progressive cumulative X-ray irradiation, and consists of cells and / or their progeny cells with a stable radioresistant phenotype; wherein the stable radioresistant phenotype can be stably passaged for at least 10 generations under maintenance irradiation of 2 Gy per week.

3. The method for constructing the human cervical cancer radiotherapy-resistant cell line ME180-R and maintaining stable radioresistance phenotype as described in claim 1, characterized in that: Includes the following steps: S1: Initial cell culture: Obtain and culture human cervical cancer cells ME180; S2: Induction irradiation: When the human cervical cancer cells ME180 are in the exponential growth phase, fractionated X-ray irradiation is started, with a single irradiation dose of 2 Gy every 72 hours, for a cumulative 40 irradiations, and a total dose of 80 Gy is obtained to obtain the human cervical cancer radiotherapy resistant cell line ME180-R. S3: Maintenance irradiation: After the human cervical cancer radioresistant cell line ME180-R obtained in step S2 is passaged for 2-3 generations, X-ray maintenance irradiation is started at a frequency of 2 Gy once a week to maintain the stability of its radioresistant phenotype.

4. The method of claim 3, wherein: In steps S2 and S3, the irradiation is performed using 6MV X-rays generated by a linear accelerator at a dose rate of 300 cGy / min.

5. The method of claim 3, wherein: During the irradiation and culture in steps S2 and / or S3, cell digestion and passage are performed when the cell confluence reaches 90%.

6. The method of claim 3, wherein: The method also comprises a step of performing a multi-dimensional radiosensitivity evaluation of the cell line obtained to quantify its radioresistant phenotype, said evaluation comprising the determination of its mean lethal dose D0, the threshold dose Dq, the survival fraction SF2 and the radioresistance multiplication factor. q , the survival fraction SF2 and the radioresistance multiplication factor.

7. The use of the human cervical cancer radiotherapy resistant cell line ME180-R as described in claim 1 in the preparation of preclinical research models, reagents, or kits for screening and / or evaluating cervical cancer radiosensitizers.

8. The use of the human cervical cancer radiotherapy resistant cell line ME180-R as described in claim 1 in the preparation of preclinical research models, reagents, or kits for studying the molecular mechanisms of cell survival regulation related to radiotherapy resistance in cervical cancer.

9. A kit for assessing radiosensitivity of cervical cancer cells, characterized by: The kit contains the human cervical cancer radiotherapy resistant cell line ME180-R as described in claim 1.

10. A preclinical evaluation system for screening radiosensitizers, characterized by: The system comprises the human cervical cancer radiotherapy resistant cell line ME180-R as described in claim 1.

Images